RESUMO
Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.
Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Endorribonucleases/química , Endorribonucleases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Retina/metabolismo , Ribonucleases/antagonistas & inibidoresRESUMO
The secretory capacity of a cell is constantly challenged by physiological demands and pathological perturbations. To adjust and match the protein-folding capacity of the endoplasmic reticulum (ER) to changing secretory needs, cells employ a dynamic intracellular signaling pathway known as the unfolded protein response (UPR). Homeostatic activation of the UPR enforces adaptive programs that modulate and augment key aspects of the entire secretory pathway, whereas maladaptive UPR outputs trigger apoptosis. Here, we discuss recent advances into how the UPR integrates information about the intensity and duration of ER stress stimuli in order to control cell fate. These findings are timely and significant because they inform an evolving mechanistic understanding of a wide variety of human diseases, including diabetes mellitus, neurodegeneration, and cancer, thus opening up the potential for new therapeutic modalities to treat these diverse diseases.
Assuntos
Linhagem da Célula/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Fator 6 Ativador da Transcrição/metabolismo , Animais , Apoptose , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Homeostase , Humanos , Modelos Biológicos , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Via Secretória/fisiologia , Transdução de Sinais , eIF-2 Quinase/metabolismoRESUMO
During endoplasmic reticulum (ER) stress, homeostatic signaling through the unfolded protein response (UPR) augments ER protein-folding capacity. If homeostasis is not restored, the UPR triggers apoptosis. We found that the ER transmembrane kinase/endoribonuclease (RNase) IRE1alpha is a key component of this apoptotic switch. ER stress induces IRE1alpha kinase autophosphorylation, activating the RNase to splice XBP1 mRNA and produce the homeostatic transcription factor XBP1s. Under ER stress--or forced autophosphorylation--IRE1alpha's RNase also causes endonucleolytic decay of many ER-localized mRNAs, including those encoding chaperones, as early events culminating in apoptosis. Using chemical genetics, we show that kinase inhibitors bypass autophosphorylation to activate the RNase by an alternate mode that enforces XBP1 splicing and averts mRNA decay and apoptosis. Alternate RNase activation by kinase-inhibited IRE1alpha can be reconstituted in vitro. We propose that divergent cell fates during ER stress hinge on a balance between IRE1alpha RNase outputs that can be tilted with kinase inhibitors to favor survival.
Assuntos
Endorribonucleases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células/metabolismo , Retículo Endoplasmático/metabolismo , Insulina/genética , Complexos Multienzimáticos , Dobramento de Proteína , Proteínas Serina-Treonina Quinases , Estabilidade de RNA , Ratos , RibonucleasesRESUMO
The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing ß-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory 'sweet spot', achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.
Assuntos
Trifosfato de Adenosina/farmacologia , Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/química , Endorribonucleases/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Desdobramento de Proteína/efeitos dos fármacosRESUMO
Disruption of protein folding in the endoplasmic reticulum (ER) causes unfolded proteins to accumulate, triggering the unfolded protein response (UPR). UPR outputs in turn decrease ER unfolded proteins to close a negative feedback loop. However, because it is infeasible to directly measure the concentration of unfolded proteins in vivo, cells are generically described as experiencing "ER stress" whenever the UPR is active. Because ER redox potential is optimized for oxidative protein folding, we reasoned that measureable redox changes should accompany unfolded protein accumulation. To test this concept, we employed fluorescent protein reporters to dynamically measure ER redox status and UPR activity in single cells. Using these tools, we show that diverse stressors, both experimental and physiological, compromise ER protein oxidation when UPR-imposed homeostatic control is lost. Using genetic analysis we uncovered redox heterogeneities in isogenic cell populations, and revealed functional interlinks between ER protein folding, modification, and quality control systems.
Assuntos
Retículo Endoplasmático/fisiologia , Dobramento de Proteína , Saccharomyces cerevisiae/citologia , Proteínas de Fluorescência Verde/metabolismo , Oxirredução , Saccharomyces cerevisiae/fisiologia , Estresse FisiológicoRESUMO
After lung injury, damage-associated transient progenitors (DATPs) emerge, representing a transitional state between injured epithelial cells and newly regenerated alveoli. DATPs express profibrotic genes, suggesting that they might promote idiopathic pulmonary fibrosis (IPF). However, the molecular pathways that induce and/or maintain DATPs are incompletely understood. Here we show that the bifunctional kinase/RNase-IRE1α-a central mediator of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress is a critical promoter of DATP abundance and function. Administration of a nanomolar-potent, monoselective kinase inhibitor of IRE1α (KIRA8)-or conditional epithelial IRE1α gene knockout-both reduce DATP cell number and fibrosis in the bleomycin model, indicating that IRE1α cell-autonomously promotes transition into the DATP state. IRE1α enhances the profibrotic phenotype of DATPs since KIRA8 decreases expression of integrin αvß6, a key activator of transforming growth factor ß (TGF-ß) in pulmonary fibrosis, corresponding to decreased TGF-ß-induced gene expression in the epithelium and decreased collagen accumulation around DATPs. Furthermore, IRE1α regulates DNA damage response (DDR) signaling, previously shown to promote the DATP phenotype, as IRE1α loss-of-function decreases H2AX phosphorylation, Cdkn1a (p21) expression, and DDR-associated secretory gene expression. Finally, KIRA8 treatment increases the differentiation of Krt19CreERT2-lineage-traced DATPs into type 1 alveolar epithelial cells after bleomycin injury, indicating that relief from IRE1α signaling enables DATPs to exit the transitional state. Thus, IRE1α coordinates a network of stress pathways that conspire to entrap injured cells in the DATP state. Pharmacological blockade of IRE1α signaling helps resolve the DATP state, thereby ameliorating fibrosis and promoting salutary lung regeneration.
Assuntos
Endorribonucleases , Fibrose Pulmonar Idiopática , Apoptose/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such "ER stress," we employed an ER-targeted, redox-responsive, green fluorescent protein-eroGFP-that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to "reflux" back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.
Assuntos
Citosol/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Homeostase/fisiologia , Oxirredução , Dobramento de Proteína , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Inositol-requiring enzyme 1α (IRE1α), along with protein kinase R-like endoplasmic reticulum kinase (PERK), is a principal regulator of the unfolded protein response (UPR). Recently, the 'mono'-specific IRE1α inhibitor, kinase-inhibiting RNase attenuator 6 (KIRA6), demonstrated a promising effect against multiple myeloma (MM). Side-stepping the clinical translation, a detailed UPR phenotype in patients with MM and the mechanisms of how KIRA8 works in MM remains unclear. METHODS: We characterized UPR phenotypes in the bone marrow of patients with newly diagnosed MM. Then, in human MM cells we analyzed the possible anti-tumor mechanisms of KIRA8 and a Food and Drug Administration (FDA)-approved drug, nilotinib, which we recently identified as having a strong inhibitory effect against IRE1α activity. Finally, we performed an RNA-sequence analysis to detect key IRE1α-related molecules against MM. RESULTS: We illustrated the dominant induction of adaptive UPR markers under IRE1α over the PERK pathway in patients with MM. In human MM cells, KIRA8 decreased cell viability and induced apoptosis, along with the induction of C/EBP homologous protein (CHOP); its combination with bortezomib exhibited more anti-myeloma effects than KIRA8 alone. Nilotinib exerted a similar effect compared with KIRA8. RNA-sequencing identified Polo-like kinase 2 (PLK2) as a KIRA8-suppressed gene. Specifically, the IRE1α overexpression induced PLK2 expression, which was decreased by KIRA8. KIRA8 and PLK2 inhibition exerted anti-myeloma effects with apoptosis induction and the regulation of cell proliferation. Finally, PLK2 was pathologically confirmed to be highly expressed in patients with MM. CONCLUSION: Dominant activation of adaptive IRE1α was established in patients with MM. Both KIRA8 and nilotinib exhibited anti-myeloma effects, which were enhanced by bortezomib. Adaptive IRE1α signaling and PLK2 could be potential therapeutic targets and biomarkers in MM.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Endorribonucleases/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Adulto , Idoso , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Movimento Celular , Proliferação de Células , Estudos Transversais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Prognóstico , Pirazinas/administração & dosagem , Pirimidinas/administração & dosagem , Estudos Retrospectivos , Células Tumorais CultivadasRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1003576.].
RESUMO
The inability of cells to properly fold, modify and assemble secretory and transmembrane proteins leads to accumulation of misfolded proteins in the endoplasmic reticulum (ER). Under these conditions of 'ER stress', cell survival depends on homeostatic benefits from an intracellular signaling pathway called the unfolded protein response (UPR). When activated, the UPR induces transcriptional and translational programs that restore ER homeostasis. However, under high-level or chronic ER stress, these adaptive changes ultimately become overshadowed by alternative 'terminal UPR' signals that actively commit cells to degeneration, culminating in programmed cell death. Chronic ER stress and maladaptive UPR signaling are implicated in the etiology and pathogenesis of myriad human diseases. Naturally, this has generated widespread interest in targeting key nodal components of the UPR as therapeutic strategies. Here we summarize the state of this field with emphasis placed on two of the master UPR regulators, PERK and IRE1, which are both capable of being drugged with small molecules.
Assuntos
Retículo Endoplasmático/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismo , Animais , Retículo Endoplasmático/química , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Estresse do Retículo Endoplasmático , Homeostase , Humanos , Transdução de Sinais , eIF-2 Quinase/genéticaRESUMO
Pseudomonas aeruginosa infections are associated with high mortality rates and occur in diverse conditions including pneumonias, cystic fibrosis and neutropenia. Quorum sensing, mediated by small molecules including N-(3-oxo-dodecanoyl) homoserine lactone (C12), regulates P. aeruginosa growth and virulence. In addition, host cell recognition of C12 initiates multiple signalling responses including cell death. To gain insight into mechanisms of C12-mediated cytotoxicity, we studied the role of endoplasmic reticulum stress in host cell responses to C12. Dramatic protection against C12-mediated cell death was observed in cells that do not produce the X-box binding protein 1 transcription factor (XBP1s). The leucine zipper and transcriptional activation motifs of XBP1s were sufficient to restore C12-induced caspase activation in XBP1s-deficient cells, although this polypeptide was not transcriptionally active. The XBP1s polypeptide also regulated caspase activation in cells stimulated with N-(3-oxo-tetradecanoyl) homoserine lactone (C14), produced by Yersinia enterolitica and Burkholderia pseudomallei, and enhanced homoserine lactone-mediated caspase activation in the presence of endogenous XBP1s. In C12-tolerant cells, responses to C12 including phosphorylation of p38 MAPK and eukaryotic initiation factor 2α were conserved, suggesting that C12 cytotoxicity is not heavily dependent on these pathways. In summary, this study reveals a novel and unconventional role for XBP1s in regulating host cell cytotoxic responses to bacterial acyl homoserine lactones.
Assuntos
4-Butirolactona/análogos & derivados , Apoptose , Citotoxinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Pseudomonas aeruginosa/metabolismo , Fatores de Transcrição/metabolismo , 4-Butirolactona/genética , 4-Butirolactona/metabolismo , Animais , Caspases/genética , Caspases/metabolismo , Citotoxinas/genética , Proteínas de Ligação a DNA/genética , Ativação Enzimática/genética , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Proteína 1 de Ligação a X-Box , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Under endoplasmic reticulum stress, unfolded protein accumulation leads to activation of the endoplasmic reticulum transmembrane kinase/endoRNase (RNase) IRE1α. IRE1α oligomerizes, autophosphorylates and initiates splicing of XBP1 mRNA, thus triggering the unfolded protein response (UPR). Here we show that IRE1α's kinase-controlled RNase can be regulated in two distinct modes with kinase inhibitors: one class of ligands occupies IRE1α's kinase ATP-binding site to activate RNase-mediated XBP1 mRNA splicing even without upstream endoplasmic reticulum stress, whereas a second class can inhibit the RNase through the same ATP-binding site, even under endoplasmic reticulum stress. Thus, alternative kinase conformations stabilized by distinct classes of ATP-competitive inhibitors can cause allosteric switching of IRE1α's RNase--either on or off. As dysregulation of the UPR has been implicated in a variety of cell degenerative and neoplastic disorders, small-molecule control over IRE1α should advance efforts to understand the UPR's role in pathophysiology and to develop drugs for endoplasmic reticulum stress-related diseases.
Assuntos
Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Catálise , Células Cultivadas , Reagentes de Ligações Cruzadas , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Conformação Molecular , Mutação/genética , Mutação/fisiologia , Fosforilação , Splicing de RNA/efeitos dos fármacos , Fatores de Transcrição de Fator Regulador X , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteína 1 de Ligação a X-BoxRESUMO
The endoplasmic reticulum (ER) performs a critical role in the oxidative folding of nascent proteins, such that perturbations to ER homeostasis may lead to protein misfolding and subsequent pathological processes. Among the mechanisms for maintaining ER homeostasis is a redox regulation, which is a critical determinant of the fate of ER-stressed cells. Here, we report the establishment of a system for monitoring the ER redox state in mammalian cells. The new ER redox-sensing system was developed based on the previously described monitoring system in yeast. Our system could successfully monitor the dynamic ER redox state in mammalian cells. Using this system, we find that manipulation of ER oxidases changes the ER redox state. The mammalian ER redox-sensing system could be used to study the mechanisms of ER redox regulation and provide a foundation for an approach to develop novel therapeutic modalities for human diseases related to dysregulated ER homeostasis including diabetes, neurodegeneration, and Wolfram syndrome.
Assuntos
Sistemas Computacionais , Retículo Endoplasmático/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus/metabolismo , Estresse do Retículo Endoplasmático , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Homeostase , Humanos , Camundongos , Degeneração Neural/metabolismo , Oxirredução , Dobramento de Proteína , Ratos , Proteínas Recombinantes/metabolismo , Síndrome de Wolfram/metabolismoRESUMO
Purpose: The unfolded protein response (UPR) is triggered when the protein folding capacity of the endoplasmic reticulum (ER) is overwhelmed and misfolded proteins accumulate in the ER, a condition referred to as ER stress. IRE1α is an ER-resident protein that plays major roles in orchestrating the UPR. Several lines of evidence implicate the UPR and its transducers in neurodegenerative diseases, including retinitis pigmentosa (RP), a group of inherited diseases that cause progressive dysfunction and loss of rod and cone photoreceptors. This study evaluated the contribution of IRE1α to photoreceptor development, homeostasis, and degeneration. Methods: We used a conditional gene targeting strategy to selectively inactivate Ire1α in mouse rod photoreceptors. We used a combination of optical coherence tomography (OCT) imaging, histology, and electroretinography (ERG) to assess longitudinally the effect of IRE1α deficiency in retinal development and function. Furthermore, we evaluated the IRE1α-deficient retina responses to tunicamycin-induced ER stress and in the context of RP caused by the rhodopsin mutation RhoP23H. Results: OCT imaging, histology, and ERG analyses did not reveal abnormalities in IRE1α-deficient retinas up to 3 months old. However, by 6 months of age, the Ire1α mutant animals showed reduced outer nuclear layer thickness and deficits in retinal function. Furthermore, conditional inactivation of Ire1α in rod photoreceptors accelerated retinal degeneration caused by the RhoP23H mutation. Conclusions: These data suggest that IRE1α is dispensable for photoreceptor development but important for photoreceptor homeostasis in aging retinas and for protecting against ER stress-mediated photoreceptor degeneration.
Assuntos
Degeneração Retiniana , Retinose Pigmentar , Animais , Camundongos , Envelhecimento , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Degeneração Retiniana/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Resposta a Proteínas não Dobradas , Estresse do Retículo EndoplasmáticoRESUMO
BACKGROUND: Stress of the endoplasmic reticulum (ER) leading to activation of the unfolded protein response (UPR) and alveolar epithelial cell (AEC) apoptosis may play a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our objectives were to determine whether circulating caspase-cleaved cytokeratin-18 (cCK-18) is a marker of AEC apoptosis in IPF, define the relationship of cCK-18 with activation of the UPR, and assess its utility as a diagnostic biomarker. METHODS: IPF and normal lung tissues were stained with the antibody (M30) that specifically binds cCK-18. The relationship between markers of the UPR and cCK-18 was determined in AECs exposed in vitro to thapsigargin to induce ER stress. cCK-18 was measured in serum from subjects with IPF, hypersensitivity pneumonitis (HP), nonspecific interstitial pneumonia (NSIP), and control subjects. RESULTS: cCK-18 immunoreactivity was present in AECs of IPF lung, but not in control subjects. Markers of the UPR (phosphorylated IRE-1α and spliced XBP-1) were more highly expressed in IPF type II AECs than in normal type II AECs. Phosphorylated IRE-1α and cCK-18 increased following thapsigargin-induced ER stress. Serum cCK-18 level distinguished IPF from diseased and control subjects. Serum cCK-18 was not associated with disease severity or outcome. CONCLUSIONS: cCK-18 may be a marker of AEC apoptosis and UPR activation in patients with IPF. Circulating levels of cCK-18 are increased in patients with IPF and cCK-18 may be a useful diagnostic biomarker.
Assuntos
Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/metabolismo , Queratina-18/metabolismo , Idoso , Apoptose , Biomarcadores/metabolismo , Estudos de Casos e Controles , Estresse do Retículo Endoplasmático , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Fibrose Pulmonar Idiopática/patologia , Masculino , Pessoa de Meia-Idade , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologiaRESUMO
Our recent studies indicate that endoplasmic reticulum (ER) stress causes INS-1 cell apoptosis by a Ca(2+)-independent phospholipase A(2) (iPLA(2)beta)-mediated mechanism that promotes ceramide generation via sphingomyelin hydrolysis and subsequent activation of the intrinsic pathway. To elucidate the association between iPLA(2)beta and ER stress, we compared beta-cell lines generated from wild type (WT) and Akita mice. The Akita mouse is a spontaneous model of ER stress that develops hyperglycemia/diabetes due to ER stress-induced beta-cell apoptosis. Consistent with a predisposition to developing ER stress, basal phosphorylated PERK and activated caspase-3 are higher in the Akita cells than WT cells. Interestingly, basal iPLA(2)beta, mature SREBP-1 (mSREBP-1), phosphorylated Akt, and neutral sphingomyelinase (NSMase) are higher, relative abundances of sphingomyelins are lower, and mitochondrial membrane potential (DeltaPsi) is compromised in Akita cells, in comparison with WT cells. Exposure to thapsigargin accelerates DeltaPsi loss and apoptosis of Akita cells and is associated with increases in iPLA(2)beta, mSREBP-1, and NSMase in both WT and Akita cells. Transfection of Akita cells with iPLA(2)beta small interfering RNA, however, suppresses NSMase message, DeltaPsi loss, and apoptosis. The iPLA(2)beta gene contains a sterol-regulatory element, and transfection with a dominant negative SREBP-1 reduces basal mSREBP-1 and iPLA(2)beta in the Akita cells and suppresses increases in mSREBP-1 and iPLA(2)beta due to thapsigargin. These findings suggest that ER stress leads to generation of mSREBP-1, which can bind to the sterol-regulatory element in the iPLA(2)beta gene to promote its transcription. Consistent with this, SREBP-1, iPLA(2)beta, and NSMase messages in Akita mouse islets are higher than in WT islets.
Assuntos
Diabetes Mellitus/etiologia , Retículo Endoplasmático/patologia , Fosfolipases A2 Independentes de Cálcio/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/fisiologia , Transcrição Gênica , Animais , Apoptose , Sítios de Ligação , Humanos , Células Secretoras de Insulina , Camundongos , Camundongos Transgênicos , Ligação Proteica , Estresse FisiológicoRESUMO
Increased levels of unfolded proteins in the endoplasmic reticulum (ER) of all eukaryotes trigger the unfolded protein response (UPR). Lower eukaryotes solely use an ancient UPR mechanism, whereby they up-regulate ER-resident chaperones and other enzymatic activities to augment protein folding and enhance degradation of misfolded proteins. Metazoans have evolved an additional mechanism through which they attenuate translation of secretory pathway proteins by activating the ER protein kinase PERK. In mammalian professional secretory cells such as insulin-producing pancreatic beta-cells, PERK is highly abundant and crucial for proper functioning of the secretory pathway. Through a modeling approach, we propose explanations for why a translation attenuation (TA) mechanism may be critical for beta-cells, but is less important in nonsecretory cells and unnecessary in lower eukaryotes such as yeast. We compared the performance of a model UPR, both with and without a TA mechanism, by monitoring 2 variables: (i) the maximal increase in ER unfolded proteins during a response, and (ii) the accumulation of chaperones between 2 consecutive pulses of stress. We found that a TA mechanism is important for minimizing these 2 variables when the ER is repeatedly subjected to transient unfolded protein stresses and when it sustains a large flux of secretory pathway proteins which are both conditions encountered physiologically by pancreatic beta-cells. Low expression of PERK in nonsecretory cells, and its absence in yeast, can be rationalized by lower trafficking of secretory proteins through their ERs.
Assuntos
Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica , Modelos Químicos , Biossíntese de Proteínas , Animais , Células Secretoras de Insulina/metabolismo , Chaperonas Moleculares/biossíntese , Transporte Proteico , Proteínas/metabolismo , Especificidade da Espécie , Leveduras/metabolismo , eIF-2 QuinaseRESUMO
AIMS/INTRODUCTION: Under irremediable endoplasmic reticulum (ER) stress, hyperactivated inositol-requiring enzyme 1α (IRE1α) triggers the terminal unfolded protein response (T-UPR), causing crucial cell dysfunction and apoptosis. We hypothesized that nicotinic acetylcholine receptor (nAChR) signaling regulates IRE1α activation to protect ß-cells from the T-UPR under ER stress. MATERIALS AND METHODS: The effects of nicotine on IRE1α activation and key T-UPR markers, thioredoxin-interacting protein and insulin/proinsulin, were analyzed by real-time polymerase chain reaction and western blotting in rat INS-1 and human EndoC-ßH1 ß-cell lines. Doxycycline-inducible IRE1α overexpression or ER stress agents were used to induce IRE1α activation. An α7 subunit-specific nAChR agonist (PNU-282987) and small interfering ribonucleic acid for α7 subunit-specific nAChR were used to modulate nAChR signaling. RESULTS: Nicotine inhibits the increase in thioredoxin-interacting protein and the decrease in insulin 1/proinsulin expression levels induced by either forced IRE1α hyperactivation or ER stress agents. Nicotine attenuated X-box-binding protein-1 messenger ribonucleic acid site-specific splicing and IRE1α autophosphorylation induced by ER stress. Furthermore, PNU-282987 attenuated T-UPR induction by either forced IRE1α activation or ER stress agents. The effects of nicotine on attenuating thioredoxin-interacting protein and preserving insulin 1 expression levels were attenuated by pharmacological and genetic inhibition of α7 nAChR. Finally, nicotine suppressed apoptosis induced by either forced IRE1α activation or ER stress agents. CONCLUSIONS: Our findings suggest that nAChR signaling regulates IRE1α activation to protect ß-cells from the T-UPR and apoptosis under ER stress partly through α7 nAChR. Targeting nAChR signaling to inhibit the T-UPR cascade may therefore hold therapeutic promise by thwarting ß-cell death in diabetes.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Nicotínicos/metabolismo , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Apoptose/fisiologia , Linhagem Celular , Humanos , Células Secretoras de Insulina/metabolismo , Substâncias Protetoras/farmacologia , RatosRESUMO
BACKGROUND: Myriad challenges to the proper folding and structural maturation of secretory pathway client proteins in the endoplasmic reticulum (ER) - a condition referred to as "ER stress" - activate intracellular signaling pathways termed the unfolded protein response (UPR). SCOPE OF REVIEW: Through executing transcriptional and translational programs the UPR restores homeostasis in those cells experiencing manageable levels of ER stress. But the UPR also actively triggers cell degeneration and apoptosis in those cells that are encountering ER stress levels that exceed irremediable thresholds. Thus, UPR outputs are "double-edged". In pancreatic islet ß-cells, numerous genetic mutations affecting the balance between these opposing UPR functions cause diabetes mellitus in both rodents and humans, amply demonstrating the principle that the UPR is critical for the proper functioning and survival of the cell. MAJOR CONCLUSIONS: Specifically, we have found that the UPR master regulator IRE1α kinase/endoribonuclease (RNase) triggers apoptosis, ß-cell degeneration, and diabetes, when ER stress reaches critical levels. Based on these mechanistic findings, we find that novel small molecule compounds that inhibit IRE1α during such "terminal" UPR signaling can spare ER stressed ß-cells from death, perhaps affording future opportunities to test new drug candidates for disease modification in patients suffering from diabetes.
Assuntos
Diabetes Mellitus/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas , Animais , HumanosRESUMO
Abnormally high levels of misfolded proteins in the endoplasmic reticulum (ER) lumen result in a stress state that contributes to the progression of several pathological conditions including diabetes, cancer, neurodegeneration, and immune dysregulation. ER stress triggers a dynamic signaling pathway known as the unfolded protein response (UPR). The UPR enforces adaptive or cell death programs by integrating information about the intensity and duration of the stress stimuli. Thus, depending on the disease context, ER stress signaling can be beneficial or detrimental. We discuss current efforts to develop small molecules to target distinct components of the UPR, and their possible applications in treating human disease, focusing on neurodegenerative diseases, metabolic disorders, and cancer.